Libraries of diverse small molecules are important to probe and drug discovery. The current trend toward building massive screening collections to support drug development, a special application of chemical biology, can limit their broader potential. Biology-driven construction methods (Wallace et al., 2011) are rapidly emerging to bring chemical libraries back on a viable path.

Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.

BACKGROUND: The Blue Obelisk movement was established in 2005 as a response to the lack of Open Data, Open Standards and Open Source (ODOSOS) in chemistry. It aims to make it easier to carry out chemistry research by promoting interoperability between chemistry software, encouraging cooperation between Open Source developers, and developing community resources and Open Standards. RESULTS: This contribution looks back on the work carried out by the Blue Obelisk in the past 5 years and surveys progress and remaining challenges in the areas of Open Data, Open Standards, and Open Source in chemistry. CONCLUSIONS: We show that the Blue Obelisk has been very successful in bringing together researchers and developers with common interests in ODOSOS, leading to development of many useful resources freely available to the chemistry community.

Combination therapies that enhance efficacy or permit reduced dosages to be administered have seen great success in a variety of therapeutic applications. More fundamentally, the discovery of epistatic pathway interactions not only informs pharmacologic intervention but can be used to better understand the underlying biological system. There is, however, no systematic and efficient method to identify interacting activities as candidates for combination therapy and, in particular, to identify those with synergistic activities. We devised a pooled, self-deconvoluting screening paradigm for the efficient comprehensive interrogation of all pairs of compounds in 1000-compound libraries. We demonstrate the power of the method to recover established synergistic interactions between compounds. We then applied this approach to a cell-based screen for anti-inflammatory activities using an assay for lipopolysaccharide/interferon-induced acute phase response of a monocytic cell line. The described method, which is >20 times as efficient as a naïve approach, was used to test all pairs of 1027 bioactive compounds for interleukin-6 suppression, yielding 11 pairs of compounds that show synergy. These 11 pairs all represent the same two activities: β-adrenergic receptor agonists and phosphodiesterase-4 inhibitors. These activities both act through cyclic AMP elevation and are known to be anti-inflammatory alone and to synergize in combination. Thus we show proof of concept for a robust, efficient technique for the identification of synergistic combinations. Such a tool can enable qualitatively new scales of pharmacological research and chemical genetics.

The enzymes that regulate histone methylation states and the protein domains that recognize methylated histone residues have been implicated in a number of human diseases, including cancer, as a result of their ability to affect transcriptional changes by altering chromatin structure. These proteins are recognized as potential therapeutic targets for the treatment of diseases associated with epigenetic disruption; however, few inhibitors of their activity have been identified. The majority of histone demethylase and methyltransferase enzyme inhibitors have been discovered on the basis of their structural similarity to substrates or known inhibitors of enzymes with analogous mechanisms. The general lack of potency and specificity of these compounds indicates that novel chemotypes are needed to address the large number of recently discovered histone-modifying enzymes. High-throughput screening (HTS) allows rapid testing of chemically diverse small molecule libraries, provided assays amenable to HTS exist. Here we review the biochemical and cellular assays available for testing the proteins and enzymes that regulate histone methylation. Progress in the development of high-throughput, sensitive, and robust assays will enable discovery of small molecules for epigenetic therapy.

Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described.

Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC(50) values for inhibition of APE1 incision of depurinated plasmid DNA by lucanthone and hycanthone were 5 µM and 80 nM, respectively. The K(D) values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e.g., TRIS and DMSO, suggesting that the mechanism of APE1 breakdown may involve free radical-induced peptide bond cleavage.

The human cytochrome P450 (CYP450) isozymes are the most important enzymes in the body to metabolize many endogenous and exogenous substances including environmental toxins and therapeutic drugs. Any unnecessary interactions between a small molecule and CYP450 isozymes may raise a potential to disarm the integrity of the protection. Accurately predicting the potential interactions between a small molecule and CYP450 isozymes is highly desirable for assessing the metabolic stability and toxicity of the molecule. The National Institutes of Health Chemical Genomics Center (NCGC) has screened a collection of over 17,000 compounds against the five major isozymes of CYP450 (1A2, 2C9, 2C19, 2D6, and 3A4) in a quantitative high throughput screening (qHTS) format. In this study, we developed support vector classification (SVC) models for these five isozymes using a set of customized generic atom types. The CYP450 data sets were randomly split into equal-sized training and test sets. The optimized SVC models exhibited high predictive power against the test sets for all five CYP450 isozymes with accuracies of 0.93, 0.89, 0.89, 0.85, and 0.87 for 1A2, 2C9, 2C19, 2D6, and 3A4, respectively, as measured by the area under the receiver operating characteristic (ROC) curves. The important atom types and features extracted from the five models are consistent with the structural preferences for different CYP450 substrates reported in the literature. We also identified novel features with significant discerning power to separate CYP450 actives from inactives. These models can be useful in prioritizing compounds in a drug discovery pipeline or recognizing the toxic potential of environmental chemicals.

A December 2010 meeting, "Down Syndrome: National Conference on Patient Registries, Research Databases, and Biobanks," was jointly sponsored by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) at the National Institutes of Health (NIH) in Bethesda, MD, and the Global Down Syndrome Foundation (GDSF)/Linda Crnic Institute for Down Syndrome based in Denver, CO. Approximately 70 attendees and organizers from various advocacy groups, federal agencies (Centers for Disease Control and Prevention, and various NIH Institutes, Centers, and Offices), members of industry, clinicians, and researchers from various academic institutions were greeted by Drs. Yvonne Maddox, Deputy Director of NICHD, and Edward McCabe, Executive Director of the Linda Crnic Institute for Down Syndrome. They charged the participants to focus on the separate issues of contact registries, research databases, and biobanks through both podium presentations and breakout session discussions. Among the breakout groups for each of the major sessions, participants were asked to generate responses to questions posed by the organizers concerning these three research resources as they related to Down syndrome and then to report back to the group at large with a summary of their discussions. This report represents a synthesis of the discussions and suggested approaches formulated by the group as a whole.

In an effort to develop strategies that improve the efficacy of existing anticancer agents, we have conducted a siRNA-based RNAi screen to identify genes that, when targeted by siRNA, improve the activity of the topoisomerase I (Top1) poison camptothecin (CPT). Screening was conducted using a set of siRNAs corresponding to over 400 apoptosisrelated genes in MDA-MB-231 breast cancer cells. During the course of these studies, we identified the silencing of MAP3K7 as a significant enhancer of CPT activity. Follow-up analysis of caspase activity and caspase-dependent phosphorylation of histone H2AX demonstrated that the silencing of MAP3K7 enhanced CPT-associated apoptosis. Silencing MAP3K7 also sensitized cells to additional compounds, including CPT clinical analogs. This activity was not restricted to MDA-MB-231 cells, as the silencing of MAP3K7 also sensitized the breast cancer cell line MDA-MB-468 and HCT-116 colon cancer cells. However, MAP3K7 silencing did not affect compound activity in the comparatively normal mammary epithelial cell line MCF10A, as well as some additional tumorigenic lines. MAP3K7 encodes the TAK1 kinase, an enzyme that is central to the regulation of many processes associated with the growth of cancer cells (e.g. NF- κB, JNK, and p38 signaling). An analysis of TAK1 signaling pathway members revealed that the silencing of TAB2 also sensitizes MDA-MB-231 and HCT-116 cells towards CPT. These findings may offer avenues towards lowering the effective doses of Top1 inhibitors in cancer cells and, in doing so, broaden their application.